Operaphenix imager

For nuclear γH2Ax staining, cells were seeded in 96-well plates (CellCarrier-96 Ultra Collagen-coated PerkinElmer, 6055700) with 10⁵ cells per 100 µl per well and incubated at 37 °C for 18 h. Treatment with HRO761 was performed using the HP Dispenser. After compound exposure, cells were fixed in 4% (v/v) paraformaldehyde (PFA) in PBS for 10 min at room temperature, washed twice with PBS and permeabilized/ blocked with 0.5% (v/v) Triton X-100, 0.5% BSA in PBS for 1 h at room temperature. Cells were then incubated with 1:2,000 anti-γH2AX (Millipore, 05-636) primary antibodies in blocking buffer overnight at 4 °C. The cells were then washed three times with 0.5% (v/v) Triton X-100 in PBS, followed by incubation with 1:2,000 goat anti mouse Alexa Fluor 488-conjugated antibodies (Invitrogen, A11001) and 1 µg ml⁻¹ DAPI in blocking buffer for 1 h at room temperature. Cells were then washed three times with 0.5% (v/v) Triton X-100, and 200 μl PBS was finally added to each well before imaging. Images were captured and analysed using the PerkinElmer Opera Phenix imager and Harmony 4.9 software.

Dissociated hiPSC-CMs treated with compounds were replated onto 20× FN-coated CellCarrier96 Ultra Microplates (Perkin Elmer) at 1 × 10⁴ cells/well and incubated for 4 days. Then the cells were fixed with 4% PFA and stained with cTNT (Thermo, MS-295-P, 1:500 dilution) and goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 546 (Invitrogen, A-11035, 1:500 dilution). Images were acquired using an OperaPhenix imager (Perkin Elmer, Waltham, MA). Roundness was measured using Harmony High-Content Imaging and Analysis Software (Perkin Elmer).

During the screening, images were obtained using an OperaPhenix imager (Perkin Elmer) after immunostaining. One image was acquired from each well using a ×10 air NA0.3 objective. The Hoechst fluorescence intensity was detected for the identification of the nuclei area. The cytoplasm area was defined based on the fluorescence intensity of EmGFP. The fluorescence intensity of the Alexa Fluor 647 was quantified to measure reporter protein levels in the defined cytoplasm region. Data for each treatment condition are expressed as the mean and standard deviation of the wells, which were derived from the average of individual cells in each well.